Mir363-3p Treatment Attenuates Long-Term Cognitive Deficits Precipitated by an Ischemic Stroke in Middle-Aged Female Rats.

Cognitive impairment and memory loss are commonly seen after stroke and a third of patients will develop signs of dementia a year after stroke. Despite a large number of studies on the beneficial effects of neuroprotectants, few studies have examined the effects of these compounds/interventions on long-term cognitive impairment. Our previous work showed that the microRNA mir363-3p reduced infarct volume and sensory-motor impairment in the acute stage of stroke in middle-aged females but not males. Thus, the present study determined the impact of mir363-3p treatment on stroke-induced cognitive impairment in middle-aged females. Sprague-Dawley female rats (12 months of age) were subjected to middle cerebral artery occlusion (MCAo; or sham surgery) and injected (iv) with mir363-3p mimic (MCAo + mir363-3p) or scrambled oligos (MCAo + scrambled) 4 h later. Sensory-motor performance was assessed in the acute phase (2-5 days after stroke), while all other behaviors were tested 6 months after MCAo (18 months of age). Cognitive function was assessed by the novel object recognition test (declarative memory) and the Barnes maze (spatial memory). The MCAo + scrambled group showed reduced preference for a novel object after the stroke and poor learning in the spatial memory task. In contrast, mir363-3p treated animals were similar to either their baseline performance or to the sham group. Histological analysis showed significant deterioration of specific white matter tracts due to stroke, which was attenuated in mir363-3p treated animals. The present data builds on our previous finding to show that a neuroprotectant can abrogate the long-term effects of stroke.

Reproductive Senescence and Ischemic Stroke Remodel the Gut Microbiome and Modulate the Effects of Estrogen Treatment in Female Rats.

Our previous work has shown that reproductively senescent (or middle-aged; 10-12-month-old) Sprague-Dawley female rats, that are naturally estrogen-deficient, have worse stroke outcomes as compared to normally estrous-cycling adult (5-6-month-old) females. Paradoxically, estrogen replacement to this middle-aged group exacerbates stroke outcomes, while it is neuroprotective in adult females. Recent studies reveal an important role for the gut microbiome and gut metabolites in cardiovascular health, including stroke outcomes. To determine whether gut dysbiosis underlies stroke severity in reproductive senescent females, and underlies the anomalous effects of estrogen on stroke, we compared the gut microbiota and gut metabolites pre and post stroke in (a) gonadally intact adult and middle-aged females, (b) in ovariectomized and estrogen-treated (OVX+E) adult and OVX+E middle-aged females, and (c) in middle-aged OVX+E females after fecal microbiome transfer. Our data show significant gut dysbiosis in reproductive senescent females at baseline and after stroke as indicated by an elevated ratio of the major phyla, Firmicutes/Bacteroidetes (F:B), reduced alpha diversity, and significant shifts in beta diversity as compared with adult females. Specific bacterial families were also altered as a result of reproductive aging, as well as gut metabolites, including elevated serum endotoxin levels and decreased short-chain fatty acids (SCFAs), with a concomitant increase in IL-17A, indicating that reproductive senescence significantly affects gut communities under pathologic conditions. Despite the differences in gonadally intact adult and middle-aged females, estrogen-treated ovariectomized (OVX+E) females of either age group displayed no differences in the major phyla, but there was increased abundance in specific bacterial taxa, including Prevotella and Lactobacillus. The SCFA butyrate was significantly reduced at baseline in the middle-aged OVX+E females, while circulating endotoxin LPS were elevated in this group after stroke, suggesting that gut metabolites were differently affected by estrogen treatment in the two age groups. A fecal transfer from adult OVX+E females to middle-aged OVX+E females significantly reduced infarct volume, improved behavioral recovery and transiently reduced IL-17A expression. These data provide the first evidence that microbial gut communities and metabolites are altered by reproductive senescence in female rats at baseline and after stroke, and suggest that estrogen may impact stroke recovery differently in adult and reproductive senescent females due to an age-specific effect on gut microbiota and metabolites.

Sex hormones and stroke: Beyond estrogens.

Stroke risk and poor stroke outcomes in postmenopausal women have usually beeen attributed to decreased levels of estrogen. However, two lines of evidence suggest that this hormone may not be solely responsible for elevated stroke risk in this population. First, the increased risk for CVD and stroke occurs much earlier than menopause at a time when estrogen levels are not yet reduced. Second, estrogen therapy has not successfully reduced stroke risk in all studies. Other sex hormones may therefore also contribute to stroke risk. Prior to menopause, levels of the gonadotrophin Follicle Stimulating Hormone (FSH) are elevated while levels of the gonadal peptide inhibin are lowered, indicating an overall decrease in ovarian reserve. Similarly, reduced estrogen levels at menopause significantly increase the ratio of androgens to estrogens. In view of the evidence that androgens may be unfavorable for CVD and stroke, this elevated ratio of testosterone to estrogen may also contribute to the postmenopause-associated stroke risk. This review synthesizes evidence from different clinical populations including natural menopause, surgical menopause, women on chemotherapy, and preclinical stroke models to dissect the role of ovarian hormones and stroke risk and outcomes.

Mir363-3p attenuates post-stroke depressive-like behaviors in middle-aged female rats.

Women are more likely to develop Post Stroke Depression (PSD) than men and generally do not respond well to anti-depressants with age. This study investigated the effect of microRNA mir363-3p treatment on PSD using a physiologically-relevant animal model. Our previous work showed that mir363-3p treatment, delivered post-stroke, effectively reduces infarct volume in the acute phase of stroke in middle-aged females but not males. Middle-aged female Sprague Dawley rats were tested for baseline sensory motor function and depressive-like behaviors, and then subjected to ischemic stroke via middle cerebral artery occlusion (MCAo) or sham surgery. Animals received either control oligos (MCAo+scrambled, Sham+scrambled) or mir363-3p (MCAo+mir363-3p, Sham+mir363-3p) treatment 4 h later. Sensory motor function and depressive-like behaviors were reassessed up to 100 d after stroke, and circulating levels of IL-6, TNF-alpha and Brain-Derived Neurotrophic Factor (BDNF) were quantified at regular intervals. Prior to termination, Fluorogold was injected into the striatum to assess meso-striatal projections. MCAo+scrambled animals had impaired sensorimotor performance in the acute phase (5 days) of stroke and developed anhedonia, decreased sociability and increased helplessness in the chronic phase. MCAo+mir363-3p animals showed significantly less sensory motor impairment and fewer depressive-like behaviors. IL-6 and TNF-alpha were elevated transiently at 4 weeks after MCAo in both groups. BDNF levels decreased progressively after stroke in the MCAo+scrambled group, and this was attenuated in the mir363-3p group. The number of retrogradely-labeled SNc and VTA cells was reduced in the ischemic hemisphere of the MCAo+scrambled group. In contrast, there was no interhemispheric difference in the number of retrogradely-labeled SNc and VTA cells of MCAo+mir363-3p treated animals. Our results support a therapeutic role for mir363-3p for long-term stroke disability.